Fig 1.
CPE mutation and the effect on enzymatic activity.
(A): Nucleotide and (B): amino acid alignment of WT-hCPE and a human CPE mutant identified from an EST database. The mutant was named as TC-CPE due to the T to C mutation of hCPE gene at nucleotide 980 of exon 4. C: X-ray crystal structure of the catalytic domain of duck carboxypeptidase D (CPD, Protein Data Bank ID #1qmu), a homologous protein to CPE. The TC mutation occurs in the a-helix 4of CPD (see arrow). Condition medium from (D)WT-CPE and (E) TC-CPE transfected COS-7 cells were collected. The enzymatic activity of CPE from the conditioned medium was analyzed by HPLC. Condition medium from untransfected COS-7 cells were also analyzed and found to have no CPE activity (data not shown).
Fig 2.
Over-expression of TC-CPE on neuroprotection.
(A) N2A cells were transfected with EV, WT-CPE or TC-CPE for 24 h. The qRT-PCR showed no significant differences in transcript expression between WT-CPE and TC-CPE. t-test, p>0.05, n = 4. (B) N2A cells were transfected with EV, WT-CPE or TC-CPE for 72 h. The LDH release assay shows significantly reduced LDH released from cells transfected with WT-CPE compared to the EV group, while cells expressing TC-CPE had no significant effect. One-way ANOVA analysis followed by Tukey’s multiple comparison test: F(2, 27) = 6.93, p<0.01,n = 10. (C) N2A cells were transfected with EV, WT-CPE or TC-CPE for 24 h, and then 100 μM H2O2 was added for 48h. The results from the LDH release assay showed that cells expressing WT-CPE were protected against the oxidative stress, while cells expressing TC-CPE werenot. One-way ANOVA analysis followed by Tukey’s multiple comparison test: F(2, 12) = 8.99, p<0.01, n = 5. (D) TUNEL staining of N2A cells transfected with EV, WT-CPE or TC-CPE for 24 h, and then 100 μM H2O2 was added for 48 h. (E) The bar graph represents the quantification of the dead cells as a % of the total number of cells determined by the DAPI staining. One-way ANOVA analysis followed by Tukey’s multiple comparison test: F(2, 9) = 5.581, p <0.05, n = 4.
Fig 3.
Expression and secretion of TC-CPE in N2A cells.
(A) Representative Western blot analysis of CPE expression after EV, WT-CPE or TC-CPE transfection for 24 h in N2A cells. (B) Bar graphs show the quantification of CPE expression normalized to actin. One-way ANOVA (F(2,6) = 85.04, p<0.001, n = 3) followed by Tukey’s multiple comparison test. (C) Representative Western blot analysis of secreted CPE after transfection of EV, WT-CPE or TC-CPE for 24 h in N2A cells. (D) N2A cells were treated with EV, WT-CPE or TC-CPE condition medium for 48 h in the presence of 100 μM H2O2. LDH release assay demonstrated that WT-CPE condition medium protected against oxidative stress, but not TC-CPE condition medium.One-way ANOVA analysis followed by Tukey’s multiple comparison test: F(2,12) = 5.842, p <0.05, n = 5.
Fig 4.
TC-CPE “hijacks” WT CPE in N2A cells.
(A) Representative Western blot analysis of CPE expression from cell lysates after various treatments for 24 h in N2A cells. (B) Bar graphs show the quantification of CPE expression from cell lysates normalized to actin. (C) Representative Western blot analysis of secreted CPE after various treatments for 24 h in N2A cells. (D) Bar graphs show the quantification of secreted CPE levels. For CPE secretion, one-way ANOVA (F(3,8) = 164.9, p<0.001, n = 3) followed by Tukey’s multiple comparison test. (E) N2A cells were transfected with EV, WT-CPE or WT-CPE +TC-CPE for 24 h, and then 100 μM H2O2 was added for 48 h. LDH release assay indicated that the neuroprotection of WT-CPE against oxidative stress was not affected by co-transfection with TC-CPE in N2A cells. One-way ANOVA analysis followed by Tukey’s multiple comparison test: F(2,12) = 7.139, p < 0.05, n = 5.
Fig 5.
Over-expression of TC-CPE up-regulates CHOP levels.
(A) Immunofluorescence of N2A cells expressing WT-CPE and TC-QQ. Double immunostaining of CPE (green) with calnexin (red) in N2A cells. Note that TC-CPE is co-localized with calnexin (orange) in the perinuclear area and accumulated in the ERin N2A cells, and apparent enhancement of the accumulation of TC-CPE in the ER with MG132 treatment. (B) Representative Western blot analysis of CHOP expression after EV, WT-CPE or TC-CPE transfection for 24 h in N2A cells. 10 μM tunicamycin was added to N2A cell for 24 h as positive control. (C) Bar graphs show the quantification of CHOP expression normalized to actin. One-way ANOVA (F(2,9) = 11.55, p<0.01, n = 4) followed by Tukey’s multiple comparison test.
Fig 6.
Proteasome degradation pathway of TC-CPE in N2A cells.
(A) Representative Western blot analysis of CPE protein levels in N2A cells transfected with EV, WT-CPE, TC-CPE, and treated with MG132 or without (Veh). (B) Bar graphs show fold increase (%) CPE protein normalized to actin, in treated versus untreated cells transfected with EV, WT-CPE or TC-CPE. ND: Not detectable. One-way ANOVA (F(2,15) = 14.16, p <0.001, n = 6) followed by Tukey’s multiple comparison test, *p<0.05 compared to EV. (C) Western blot analysis of secreted CPE protein levels in N2A cells transfected with WT-CPE or TC-CPE, in the presence or absence of MG132. Note that MG132 did not affect the secreted levels of TC-CPE protein in N2A cells. t-test, p > 0.05, n = 4.